An amperometric method for the rapid detection of extended-spectrum β-lactamase producing<em> Escherichia coli</em> in wastewater treatment plant effluents

National audienceContext: Extended-spectrum β-lactamase-producing Escherichia coli (ESBL E. coli) are resistant to most β-lactams and have become a major concern in human and veterinary medicine. As E. coli and antibiotic resistant strains are part of the intestinal flora of humans, large amounts of these bacteria are present in wastewaters. Though treatments are performed in wastewater treatment plants (WWTP), large quantities of bacteria are still present in the treated effluents rejected into the environment. These releases can cause contaminations of recreational waters and thus present a health risk to exposed populations. Therefore, rapid and convenient assays are highly desired for the quantification of ESBL E. coli in the wastewater network and in natural environments. Objective of the study: Development of a nitrocefin-based amperometric method for the rapid quantification of ESBL E. coli in WWTP effluents Methods: Raw and treated wastewaters were filtered in duplicate through 0.45 μm filters (HAWP, 47 mm, Millipore). The amperometric assay involved two main steps: (1) the subculturing of the filtered samples in the presence of cefotaxime supplemented or not with the potassium clavulanate (ESBL inhibitor) for a few hours (4-5h) followed by, (2) the incubation of each subculture filtrate (v = 10 mL; HVLP filter, 0.45 μm, 13 mm, Millipore) with the nitrocefin substrate which hydrolysis was monitored by amperometry. iCef and iClav correspond to the intensity of the anodic current measured (~ + 0.2 V vs. Ag/AgCl) for the sample incubated with the cefotaxime without and with potassium clavulanate, respectively. The value i = iCef – iClav was calculated and selected as the analytical response to assess the amount of EBSL E. coli producers. Results: The mean calibration plots for the raw and treated wastewaters (Figure 1) were obtained by analyzing CTX-M type ESBL E. coli strains found in wastewaters (blaCTX-M-1 and blaCTX-M-15 genes) and were used for the determination of ESBL E. coli in 20 raw wastewater and 20 treated wastewater samples. To check the reliability of the amperometric assay, the results were compared to a conventional counting on TBX agar plates supplemented with cefotaxime (Figure 2). Conclusion: An excellent correlation was obtained between the amperometric assay and the enumeration. This amperometric assay (5-6h) which is considerably less time-consuming than the culture-based method (24h) holds great promise for the rapid quantification of ESBL E. coli in the wastewater networks but also in other types of water samples (rivers, marine waters, etc.)

Détection ampérométrique des <em>Escherichia coli</em> productrices de beta-lactamases à spectre étendu dans les effluents de station d’épuration

National audienceLes Escherichia coli productrices de β-lactamases à spectre étendu (E. coli BLSE) sont résistantes à la plupart des β-lactamines et deviennent un problème majeur de santé publique en médecine humaine. Leur présence dans le tube digestif des humains fait que grandes quantités d’E. coli BLSE sont présentes dans les eaux usées qui arrivent dans les stations d’épuration (STEP). Malgré les traitements réalisés dans les STEP, des quantités non négligeables d’E. coli BLSE sont encore présentes dans les effluents traités directement rejetés dans l’environnement. Ces rejets peuvent être à l’origine d’une contamination des eaux récréatives et constituer un risque sanitaire pour les populations exposées. La mise au point de tests rapides et pratiques est donc nécessaire pour détecter et quantifier ces E. coli BLSE dans les réseaux d’eaux usées et les environnements naturels. Cette étude à eu pour objectif de développer une méthode ampérométrique rapide pour quantifier les E. coli BLSE dans les effluents de STEP. La première étape consiste en une double filtration sur membranes d’un échantillon d’eau de STEP, suivie d’une analyse ampérométrique en deux étapes : (1) une mise en culture (4-5h) de chaque membrane dans un milieu liquide contenant du céfotaxime ± acide clavulanique (inhibiteur de BLSE) et (2) une incubation (15min) de chaque culture filtrée sur membrane en présence de Nitrocéfine, dont l’hydrolyse par les β-lactamases est suivie par ampérométrie. Deux intensités sont ainsi mesurées : iCef et iClav et la valeur i = iCef - iClav est utilisée pour quantifier les E. coli BLSE présentes dans l’échantillon en se basant sur des courbes de calibration. Une bonne corrélation de cette estimation ampérométrique (5-6h) avec un dénombrement classique sur milieu gélosé (24h) est obtenue après avoir analysé une quarantaine d’échantillons. Cette méthode est très prometteuse dans le domaine de l’analyse des eaux usées et des eaux récréatives

A nitrocefin-based amperometric assay for the rapid quantification of extended-spectrum β-lactamase-producing Escherichia coli in wastewaters

International audienceA sensitive and inexpensive amperometric assay based on the electrochemical detection of the beta-lactamase activity using the nitrocefin as substrate was developed for the rapid and quantitative detection of extended spectrum beta-lactamase-producing Escherichia colt (ESBL-EC) in urban wastewaters. The specific detection of ESBL-EC was achieved by culturing the filtered sample in a medium containing the cefotaxime supplemented or not with the potassium clavulanate inhibitor. This step was followed by the incubation of each subculture filtrate with the nitrocefin substrate which hydrolysis was monitored by amperometry using disposable carbon screen-printed sensors. Current intensities i(Cef) and i(Clav) correspond to the intensity of the anodic current measured (similar to+ 0.2 V vs. Ag/AgCl) for the sample incubated with the cefotaxime without and with potassium clavulanate, respectively. The intensity value i = i(Cef) iClav was chosen as the analytical response. ESBL-EC calibration plots were established with artificially contaminated wastewater samples. This assay allowed the detection of ESBL-EC amounts as low as 10 cfu in treated effluents and 100 cfu in raw wastewaters with short time analysis of 5.5 h and 4.5 h, respectively. The amperometric method was applied to the analysis of 38 wastewater samples and the results were in good agreement with CFU counts on a selective chromogenic medium for 24 h. Owing to its rapidity, convenience, low-cost and portability, this assay is a promising tool to obtain quantitative data on antimicrobial-resistant E. coli in wastewater effluents. Furthermore, this assay might be used to improve wastewater treatment plant processes in order to minimize the release of antibiotic resistant bacteria into the aquatic environment

Rapid dissemination of Mycobacterium bovis from cattle dung to soil by the earthworm Lumbricus terrestris

International audienceIndirect transmission of Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), between wildlife and livestock is thought to occur by inhalation or ingestion of environmental substrates contaminated through animal shedding. The role of the soil fauna, such as earthworms, in the circulation of M. bovis from contaminated animal feces is of interest in the epidemiology of bTB. The objective of this study was to assess the impact of earthworm activity on M. bovis transfer from animal dung to castings and the surrounding soil. For this purpose, microcosms of soil containing the anecic eathworms Lumbricus terrestris were prepared and covered with cattle feces spiked with the M. bovis BCG strain Pasteur to carry out two separate experiments. The dissemination, the gut carriage and the excretion of M. bovis were all monitored using a specific qPCR-based assay. Our results showed that the earthworm L. terrestris was able to rapidly disseminate M. bovis from the contaminated cattle feces to the surrounding soil through casting egestion. Moreover, contaminated earthworms were shown to shed the bacteria for 4 days when transferred to a M. bovis-free soil. This study highlights for the first time the possible role of earthworms in the dissemination and the persistence of M. bovis in soils within bTB endemic areas

Rapid amperometric detection of <em>Escherichia coli</em> in wastewater by measuring beta-D glucuronidase activity with disposable carbon sensors

International audienceAn assay on the indirect amperometric quantification of the beta-D-Glucuronidase (GLUase) activity was developed for the rapid and specific detection of Escherichia coli (E. coli) in complex environmental samples. The p-aminophenyl beta-D-glucopyranoside (PAPG) was selected as an electrochemical substrate for GLUase measurement and the p-aminophenol (PAP) released during the enzymatic hydrolysis was monitored by cyclic voltammetry with disposable carbon screen-printed sensors. The intensity of the measured anodic peak current was proportional to the amount of GLUase, and therefore to the number of E. coli in the tested sample. Once the substrate concentration and pH values optimized, a GLUase detection limit of 10 ng mL(-1) was achieved. Using a procedure involving a filtration step of the bacteria followed by their incubation with the substrate solution containing both the nonionic detergent Triton X-100 as permeabilization agent and the culture media Luria broth to monitor the growth, filtered bacterial cells ranging from 5 x 10(4) to 10(8) UFC/membrane were detected within 3 h. The amperometric assay was applied to the determination of fecal contamination in raw and treated wastewater samples and it was successfully compared with conventional bacterial plating methods and uidA gene quantitative PCR. Owing to its ability to perform measurements in turbid media, the GLUase amperometric method is a reliable tool for the rapid and decentralized quantification of viable but also nonculturable E. coli in complex environmental samples